Computational protocol: Alterations in fatty acid metabolism and sirtuin signaling characterize early type‐2 diabetic hearts of fructose‐fed rats

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Protocol publication

[…] Total tissue homogenates were prepared using lysis buffer (50 mmol L−1 Tris pH 8.0, 150 mmol L−1 NaCl, 1% NP‐40, 0.1% SDS, 0.5% sodium deoxycholate) supplemented with the appropriate protease and phosphatase inhibitors. Protease and phosphatase inhibitors were also included in the solutions used in the preparation of subcellular fractionations for immunoblotting. All prepared tissue homogenates or subcellular fractions were mixed in 5X SDS loading buffer for Western blot analyses. Equal amounts of protein samples were loaded, separated by SDS‐PAGE and transferred to nitrocellulose membranes for immunoblotting with indicated antibodies. Immuno‐reactivity was visualized by an ECL reaction (GE HealthCare), and its intensity was quantified using ImageJ software (http://rsbweb.nih.gov/ij/). The following primary antibodies were sourced from (1) Cell Signaling Technology (Danvers, MA): pan‐acetyl‐lysine (#9441), phospho‐AMPKα (Thr172; #2531), AMPKα (#2532), lamin A/C (#4777), Na+/K+‐ATPase (#3010), (2) Abcam Inc. (Cambridge, MA): CD36 (#ab133625), COX IV (#ab14744), CPT1B (#ab104662), PDK4 (#ab89295), PGC1α (#ab54481), PPARα (#ab8934), α‐tubulin (#ab7291); (3) Santa Cruz Biotechnology Inc. (Dallas, TX): SIRT1 (#sc‐15404), SIRT3 (#sc‐99143); (4) Millipore (San Diego, CA): HNE‐Michael adducts (#393207); (5) Acris Antibodies (San Diego, CA): glutathione peroxidase I (GPx1; #AP32028PU‐N); (6) Thermo Scientific (Rockford): thioredoxin reductase 2 (TrxR2; #PA1‐20940). [...] SigmaPlot 13.0 (Systat Software, San Jose, CA) was used for statistical analyses. Results are presented as mean ± SE for the indicated number of independent heart experiments. Differences between control and fructose‐fed rats were evaluated by Student's t‐test or Mann–Whitney rank sum tests, as determined by data distribution. Differences are considered significant when P < 0.05. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, cryo-EM, Microscopic phenotype analysis
Organisms Rattus norvegicus
Diseases Diabetes Mellitus, Diabetes Mellitus, Type 2, Metabolic Diseases, Diabetic Cardiomyopathies
Chemicals Acyl Coenzyme A, Cardiolipins, Fructose